All phenylpropanoids are derived from cinnamic acid, which is formed from phenylalanine by the action of phenylalanine ammonia-lyase, the branch point between primary and secondary metabolism. Isoflavones represent a class of secondary metabolites produced mainly in legumes by the phenylpropanoid biosynthesis pathway. The biosynthetic pathway for free isoflavones and their relationship with several other classes of phenylpropanoids is presented in FIG. 1.
Free isoflavones rarely accumulate to high levels in soybeans. Instead they are usually conjugated to carbohydrates or organic acids. Soybean seeds contain three types of isoflavones in four different forms: the aglycones daidzein, genistein, and glycitein; the glucosides diadzin, genistin, and glycitin; the acetylglucosides 6″-O-acetyldaidzin, 6″-O-acetylgenistin, and 6″-O-acetylglycitin; and the malonylglucosides 6″-O-malonyidaidzin, 6″-O-malonylgenistin, and 6″-O-malonylglycitin. It has been reported that the isoflavones found in soybean seeds possess antihemolytic (Naim, M. et al., J. Agric. Food Chem. 24:1174–1177 (1976)), antifungal (Naim, M. et al., J. Agric. Food Chem. 22:806–810 (1974)), oestrogenic (Price, K. R. and Fenwick, G. R. Food Addit. Contam. 2:73–106 (1985)), tumor suppressing (Messina, M. and Barnes, S. J. Natl. Cancer lnst. 83:541–546 (1991); Peterson, G. et al., Biochem. Biophys. Res. Commun. 179:661–667 (1991)), hypolipidemic (Mathur, K. et al., J. Nutr. 84:201–204 (1964)), and serum cholesterol lowering (Sharma, R.D. Lipids 14:535–540 (1979)) effects. In addition, both epidemiological and dietary-intervention studies indicate that when isoflavones in soybean seeds and in the subsequent protein products made from the seeds are part of the human dietary intake, these products provide many health benefits (Messina, M. J. Am. J. Clin. Nutr. 70:439S–450S (1999)).
The content of isoflavones in soybean seeds, however, is quite variable and is affected by both genetics and environmental conditions such as growing location and temperature during seed fill (Tsukamoto, C. et al., J. Agric. Food Chem. 43:1184–1192 (1995); Wang, H. and Murphy, P. A. J. Agric. Food Chem. 42:1674–1677 (1994)). In addition, isoflavone content in legumes can be stress-induced by pathogenic attack, wounding, high UV light exposure, and pollution (Dixon, R.A. and Paiva, N.L. The Plant Cell 7:1085–1097 (1995)). To date, it has proven difficult to develop soybean lines with consistently high levels of isoflavones. Moreover, lines reported to be low in isoflavone content produced normal levels of isoflavones when grown under standard cultural conditions (Kitamura, K. et al., Jap. J. Breed. 41:651–654 (1991)).
It is well known that many nuclear protein-encoding genes belong to gene families (evolutionary-related set of genes that encode similar products). Gene families in plants can be linked or dispersed throughout the genome, lying on separate chromosomes in unrelated locations. There are several probable reasons for their dispersion (i.e., allow specialized expression and faster evolution). Soybeans are no exception and also contain gene families.
The isolation and cloning of genes associated with synthesis and metabolism of isoflavones in soybean will afford the application of molecular techniques to achieve stable, high level accumulation of isoflavones. In particular, chalcone isomerase (E.C. 5.5.1.6) which catalyzes the cyclization of chalcone (4,2′,4′,6′-tetrahydroxychalcone) and 6′-deoxychalcone (4,2′,4′-trihydroxychalcone), both of which are synthesized by the upstream enzyme chalcone synthase, into 2S)-naringenin (5,7,4′-trihydroxyflavanone) and (2S)-5-deoxyflavanone (7,4′-dihydroxyflavanone), respectfully. Since both chalcone and 6′-deoxychalcone spontaneously cyclize in solution to give enantiomeric mixtures, chalcone isomerase guarantees formation of only the biologically active (S)-isomer. A soybean chalcone isomerase has previously been identified (U.S. Pat. No. 6,054,636). The present invention provides novel chalcone isomerases from soybean (Glycine max).